Extracts of the neem tree (Azadirachta indica), the chinaberry tree (Melia azedarach), and other plants in the family Meliaceae (e.g. Azadirachta excelsa) have long been known to have insecticidal activity (Natural Pesticides from the Neem Tree, Proc. 1st Int'l Neem Conf. 1980 [H. Schmutterer et al. eds. 1982]; Natural Pesticides from the Neem Tree and Other Tropical Plants, Proc. 2nd Int'l Neem Conf. 1983 [H. Schmutterer and K. R. S. Asher eds. 1984]; Natural Pesticides from the Neem Tree and Other Tropical Plants, Proc. 3rd Int'l Neem Conf. 1986 [H. Schmutterer and K. R. S. Asher eds. 1987]). Azadirachtin, a major active ingredient in many of these extracts, is a limonoid of the tetranortriterpenoid type. Azadirachtin has been shown to be a potent insect growth regulator and feeding deterrent with value as an active ingredient in commercial insecticides (R. B. Yamasaki et al. (1987) J. Agric. Food Chem. 35:467-471).
The use of azadirachtin in commercial insecticides requires that the compound be extracted from the plant material and concentrated in a convenient form. Suitable azadirachtin concentrates are solid in form, have high levels of azadirachtin (typically greater than 1-3% by weight), and contain little or no plant oils and water. These characteristics facilitate handling, storage, distribution, and formulation.
Various methods of extracting azadirachtin from neem seeds are known in the art. J. H. Butterworth and E. D. Morgan, (1971) J. Insect. Physiol. 17:969-977, describe the extraction of azadirachtin and neem oil from neem seeds using ethanol, a common solvent for azadirachtin isolation. Further separation steps (including filtration, partition of the soluble portion between light petroleum and methanol, and chromatography of the methanol extract on Floridin earth) are used to concentrate the azadirachtin to obtain a solid. Larson (U.S. Pat. No. 4,556,562) discloses a similar method whereby neem seeds are first reduced in size to that of a regular grind of coffee. Azadirachtin is then extracted at elevated temperatures, again using ethanol. No further concentration steps are used, however, and the final extract is a liquid containing only 0.5-1% azadirachtin. Walter (U.S. Pat. No. 4,946,681) describes a related process for extracting azadirachtin from ground de-oiled neem seeds with aprotic solvents or alcohols. The liquid extract is further treated with molecular sieves to remove water. Carter et al. (U.S. Pat. No. 5,001,146) describes a two-step extraction process comprising a "cleanup" extraction with a non-polar aprotic solvent to remove the neem oil from the seeds, followed by a second extraction of the defatted neem seeds using a more polar, azadirachtin-soluble solvent. The final extract from this process is a solution containing about 4.5 g/l azadirachtin. Kleeberg (WO 92/16109) also describes a multi-step procedure whereby the azadirachtin is first extracted from neem oil and plant material using water. The aqueous fraction is then extracted with an organic solvent to remove the azadirachtin from the water. A solid azadirachtin concentrate is ultimately obtained using a slow and inefficient phase separation followed by concentration of the organic phase and crystallization.
Inefficiencies and limitations associated with the processes described above illustrate the need for more practical and versatile methods for obtaining azadirachtin concentrates. Specifically, current methods that produce extracts containing suitably high azadirachtin content (&gt;1-3% by weight) all involve expensive steps to remove neem oil, water, and other impurities. Such steps include decantation, phase separation, chromatography, crystallization, treatment with molecular sieves, and other purification steps. None of the known methods provides a suitably efficient, economical, or practical means for recovering azadirachtin in a concentrated solid form that contains little or no neem oil or water.
The present invention solves these problems by employing a simple and efficient precipitation step that produces an azadirachtin concentrate which is easily and efficiently separated from plant oils and other non-polar plant components. The present invention thus represents a significant improvement over the prior art methods.